1. Field of the Invention
The present invention relates to an assembly positionable beneath a flexible cell culture substrate membrane that supports the membrane and allows the membrane to stretch across the assembly equally in all directions when vacuum is applied at the periphery of the membrane thereby imparting equibiaxial strain to the growth surface of the membrane and to a cell culture apparatus incorporating the assembly. Alternatively, other geometries of the assembly can be employed that yield uniaxial or gradient strain in the membrane.
2. Prior Art
Elastomeric substrates have been used as surfaces for culturing cells in vitro. These substrates are usually treated to make them hydrophilic and are preferably bonded with matrix proteins. This development arose from the desire to flex cell cultures in vitro instead of culturing cells in a static environment such as exists in conventional cell culture devices. In particular, when cell cultures are grown on membranes which are flexed, the cells are strained and stressed which simulates a dynamic in vivo environment. Stressing of cells causes dramatic morphologic changes and biomechanical responses in the cells which are both long term and short term. Cell culture devices using an elastomeric substrate or a flexible cell culture membrane are flexed or stretched to induce mechanical stress and strain on the cells adhering thereto.
Such flexible cell culture membranes have been incorporated in devices by the Applicant including the single well and multi-well cell culture plates and devices disclosed in U.S. Pat. No. 4,789,601 which is incorporated herein by reference. The wells of this cell culture plate have a floor made from a flexible membrane which is treated to enable cells to adhere and grow thereon. Typically, negative pressure, from a vacuum, is applied to the underside of the cell culture plate to deform downwardly the flexible membranes in the wells of the culture plate.
An improved device is the subject of Applicant's copending U.S. patent application Ser. No. 09/201,570, filed Nov. 30, 1998 entitled "Culture Plate for Applying Mechanical Load to Cell Cultures", which is incorporated herein by reference. The device disclosed in the '570 application is similar to that of the device in the '601 patent in that it includes a multi-well culture plate having flexible cell culture membranes as the floors of the wells which may be subjected to negative pressure to downwardly flex the membrane. However, the membrane in the device in the '570 application is fixed between a base and a body so that either positive or negative pressure may be applied thereto causing upward or downward flexing of the membrane. The pressure differential applied to either of these devices may be released and reapplied to repeatedly apply stress and strain to the cells, thereby mimicking the mechanical load experienced by cells in mechanically active tissues such as the heart, lungs, skeletal muscles, bone, ligament, tendon, cartilage and the like. In this manner, the biological or biochemical responses of cells subjected to a flexed environment may be tested and studied.
However, certain cells normally experience equibiaxial strain in the plane of the cell. By equibiaxial it is meant that the strain is applied equally along both the x-axis and y-axis of the plane of the cell. In some applications of membrane stretching induced by either positive or negative pressure where the membrane is not supported, the cell culture membrane becomes arcuately shaped upon application of a differential pressure to the device. Such arcuate flexing renders difficult an analysis of the biologic responses of the cells since the strain varies in both the circumferential and radial directions. The impact of uniaxial strain in the plane of a cell (a strain in one of the x-axis or y-axis) is likewise important and difficult to analyze.
Accordingly, a need remains for a device for use with a cell culture plate with flexible cell culture membrane wells which provides for well-defined strain to be imposed on cells cultured thereon, wherein the strain is applied equibiaxially, uniaxially or in other defined strain field directions.